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Home , Camellia sinensis, Decaffeination, Tea

Journal of 2013,39(4) :420-426

A Review on of Tea Products

Zhen Wang, Qing-Sheng Li, Juan-Zhen Ni, Ci-Jie Hu, Jian-Liang Lu*

Zhejiang University Tea Research Institute, 866 Yuhangtang Road, Hangzhou 310058, PR * : corresponding author, jllu@ zju. edu. en

Abstract Since possesses some well-unknown adverse effects, many attempts have been conducted to remove it from tea products, including selective extraction with different solvents, enzymatic degradation and inhibition the caffeine synthesis pathway. Reports showed that caffeine can be safely and efficiently removed from fresh tea by hot water blanching, however this approach is not suitable for treating the rolled or dried leaves because of the unavoidable loss of . Although microwave-enhanced vacuum ice water extraction can be used for decaffeination of dried tea, time consumption seems to be the main shortage of this method. Liquefied dimethyl ether might be used as another alternative solvent for selective extraction of caffeine from tea products because of its low toxicity and residum, but its decaffeination efficiency needs to be verified furthermore. It has been proved that supercritical extraction is an effective approach for removing caffeine from different types of tea products, while the application of the technique is always limited as its expensive equipments and high running costs. Decaffeination with microbe incubation or enzymatic digestion is highly hopeful since many studies showed that some microorganisms can degrade the caffeine through induced demethylase and oxidase under gentle condition in some model systems, unfortunately, this approach is far away from application since security and quality of the decaffeinated products are rarely concerned. Low-caffeine tea products can be easily produced through normal manufacture by using the leaves harvested from some low-caffeine which are derived through genetic modification or conventional breeding, but these germplasms are very limited.

Key words Hot water blanching; dimethyl ether extraction ; SFE-C02 ; microorganisms degradation; low caffeine .

1. Introduction Tea ( sinensis) is one of the most popular beverages consumed worldwide, and it contains many physiologically active substances. The main component catechins, such as epicatechin ( EC) , pretreat ECG) , epigallocatechin ( EGC) , and ( EGCG) , exhibit various pharmacological effects, vacuum -uch as antioxidative, anti tumour, and antimicrobial activity. Also, an amino acid unique to tea, , induces a relaxing effect and potentiates the effects of anticancer . On the other hand, also contains abundant caffeine ( 1 , 3 , 7 -trimethylxanthine) , the most consumed alkaloid. It is one of the few products and the general public is readily familiar with, because of its occurrence in beverages such as tea and , as well as ,-arious soft . 2.1. 3. Caffeine has some positive physiological effects; it has been linked with enhancement of cognitive function Yu improvement of neuromuscular coordination , elevation of mood and relief of anxiety. But when the doses of caffeine produce i, high , it stimulates the central nervous, affects sleep , increases blood pressure , improves the prevalence of as follo water w rheumatoid arthritis, mutagenic and causes premature infants [ I-4 ] . Therefore, to keep the tea flavors and nutritions of low caffeine tea is welcomed by consumers. oven to Generally, the content of caffeine in dry tea is 2%-5%, in fresh tea leaves is 1. 4% 2. 8% [sJ. At present, drying 1 Wang Z et al. I Decaffeination tea producL'

the content of caffeine in decaffeinated tea is no uniform standard , countries such as Europe require caffeine content lower than 0. 5% , while China, Japan demand caffeine content lower than l 2. Approaches of decaffeination The methods of decaffeination are Water extraction, Organic solvents extraction, ( .-\C

Supercritical fluid extraction with carbon dioxide ( SFE-C02 ) , Removing caffeine with absorbents which are made of various copolymers of triallyl isocyanurate (TAlC) or vinyl acetate (VA) [?] or molecular imprinted polymers ( MIPs) [Sl , Reducing caffeine using microorganisms[9l , microwave irradiation[ IO ] , and ultrasonic devices[" ] , Ionic liquid extraction [ 12 1 etc. 2.1. Water extraction 2. 1. 1. Decaffeination with hot water At room temperature, caffeine dissolves best in chloroform 18% ( w/v) but in water the solubility is approximately 2% ( w/v). However, caffeine dissolves well in boiling water, with its solubility increasing to 70% ( w/v) at lOO"C. So hot water can be used to remove the caffeine in tea. Domestic removing caffeine with water began in 1995. Liang et al. [ 13 1 also studied the removal of caffeine in fresh tea leaves with hot water, through to the temperature, soaking time and the ratio of water to tea, the research found that 1 :20 ( w/v) of tea to water under 100"C for 3 min as the best parameters, can remove 83% of caffeine, at the same time retains 95% of catechins. Liang et al. applied for a patent that used fresh tea leaves to produce low caffeine tea powders. The method directly took the fresh tea leaves as raw material, after more than 125't steam and 100"C hot water treatment, chopped, extraction, concentration, spray drying or freeze drying to make low caffeine and powders. Hot water treatment is a convenient and inexpensive decaffeination process and leaves no organic solvent 13 1 residue. At the same time hot water plays a role of inhibiting the activities of certain [ • However, a large percentage of tea catechins was lost if rolled leaves and dry tea were decaffeinated by the hot water treatment and so the process is not suitable for processing . 2. 1. 2. Microwave-enhanced vacuum ice water extraction On the basis of the researches of hot water decaffeination , Lou et al. [ 10 1 try to remove caffeine in low temperature ( 0 "C ) , and combined microwave technique. The experiment analysis found that the removal yield of caffeine by microwave-enhanced vacuum ice water extraction ( MVIE) was 87. 6% , which was significantly higher than that by hot water extraction. Moreover, the loss of tea of green tea in the proposed method was much lower than that in the hot water extraction. The experiment was operated as follows: The dry green tea (10 g) was mixed with water ( 100 mL) and was pretreated by microwave extraction. The next step was vacuum ice water extraction, and it was carried out in a vacuum drying oven. The pretreated tea sample was put into a flask containing 100 mL of ice water (ice/water= 1: 1 , w/w). The flask was placed into the vacuum drying oven. The air in the vessel was pumped out till the degree of vacuum was 0. 1 MPa. Then the vacuum ice water extraction was performed for several hours. The optimized conditions were as follows: Solvent ( mL) to solid (g) ratio was 10: 1, microwave extraction time was 6 min, microwave power was 350 W and 2. 5 h of vacuum ice water extraction. 2. 1. 3. The application of water extraction Vuong et al. [ 14 1 attempted to develop optimal conditions for decaffeination and spray drying procedures to produce decaffeinated and high caffeine powders from green tea () . The experiment was operated as follows: Blanching the tea leaves with water at 100"C for 4 min at a water-to-tea ratio of 20: 1 ( mLI g) , then the water was decanted from the blanched tea leaves and the leaves were dried to constant weight at 70"C in a vacuum oven to give decaffeinated dried tea leaves. The decaffeinated dried tea leaves were ground, brew twice, spray drying ( 180"C for the inlet temperature and 115"C for the outlet temperature) to get the decaffeinated green tea 422 vf Journal of Tea 39 4 powers ( 7 mgl g caffeine) . 2. 2. Decaffeination using liquefied dimethyl ether Recently, attempts have been made to remove or reduce caffeine by extraction with organic solvents such as 15 1 chloroform( • However, these solvents are toxic and easily retain in food because they exist in a liquid state at room temperature. Kanda et al. (16 1 proposed an extraction technique involving the use of liquefied dimethyl ether ( DME) as an extractant. Hideki et al. (17 1 used green tea (after hot water extraction with water content of 74. 6%-76. 2%) to verify the DME extraction in both laboratory-and bench-scale processes, and then made use of high-performance liquid chromatography to measure the contents distribution of caffeine and catechins in extracted residue , orgamc extracts, and removed water. It was found that caffeine was completely removed from the green tea leaves. Approximately 25. 2% -56. 0% of catechins remained in the residue after DME extraction. In particular, 56. 0% of epigallocatechin gallate, which has the greatest activity of all catechins remained in the residue. 18 19 The characteristics of dimethyl ether decaffeination: It is very different from the existing methods( ' J , because the DME method can dewater ( dry) and extract organic extracts from biomaterials simultaneously and directly at room temperature; this means that the heating of the extractant and the downstream hot-dry process are both omitted. Furthermore, DME is a safe solvent and does not remain at room temperature; therefore, it can be used for food processing, and the organic compounds can be used for beverage production with or without pre­ treatment. The dried and decaffeinated tea residue can be used as non-caffeine tea food.

2. 3. Removal caffeine by Supercritical carbon dioxide extraction ( SFE-C02 ) The carbon dioxide decaffeination method was discovered by Kurt Zosel and patented in the early 1970s. Since then, research on decaffeination using SFE-C02 has been intensive and the resulting methods have been covered by 201 21 24 1 several patents( and reported in other publications( ' .

The principle of SFE-C02 is that pressure and temperature can influence the solubility of . At the critical temperature ( Tc) and critical pressure ( Pc) , supercritical C02 fluid has special peculiarities. Above the critical point, C02 fluid has quite a diffusion coefficient as well as gas and a low viscosity. And its density and solubility are as well as fluid. Within a certain range, there is a linear relationship between the logarithm of solubility and the logarithm of fluid density. Therefore, though controlling T and P, it can change the density, which changes the solubility of a substance, to make a selective extraction. In a supercritical state, the supercritical

C02 fluid contacts with the substance to be separated, and then extracts the different ingredients selectively according to the boiling point and molecular weight, polarity of the different ingredients. Then with decompression and warming, the supercritical C02 fluid becomes ordinary gas, so that the extracted material is completely or substantially precipitated so as to achieve the purpose of separation and purification ( 2S J .

The decaffeination of green tea using SFE-C02 is usually done by first grinding the tea leaves into small particle sizes ( < 1 mm in diameter) . The ground tea leaves are then soaked in a co-solvent (e. g. , 95: 1 , ethanol: water) , 26 1 to enhance the extraction of the caffeine[ , and are loaded into an extraction vessel that is sealed. Liquid carbon dioxide is pumped in at a designated pressure and the back pressure is continuously monitored. The liquid carbon dioxide is then heated and pumped into the extraction vessel to extract the caffeine from the ground tea leaves. The caffeine, dissolved in the supercritical carbon dioxide, is separated from the carbon dioxide and collected by a reduction in pressure which occurs in the separator sections of the SFE-C02 extractor.

It should be noted that the outcomes of the decaffeination process using SFE-C02 are influenced by vanous factors including the tea particle size, the co-solvent used to soak the tea in and the temperature, pressure and flow 27 1 rate of the C02 • Kim [ et al. using the SFE-C02 to selectively extract caffeine from green tea, found that SFE­ CO: ";th water as entrainer was more selective in green tea. Hacer Icen et al. [28 1 made use of Turkish black tea

29 1 fihe - and stalks wastes to study the effect of ethanol content on the SFE-C02 . Tu et al. [ chose the complete Wang Z et al. I Decaffeination tea products 423

1 3 -uasted leaves (produced in Lin an, Hangzhou sale) as raw materials for the study of SFE-C02 . Chen[ o] chose

1 ;reen tea, black tea, Tie Guan Yin, Pu er tea as materials for the study with SFE-C02 through single factor ~xperiment and orthogonal experiment.

The main limitation of the SFE-C02 decaffeination method is the high setup costs. However, this is outweighed .Jy several advantages such as it being a fast process with no toxic residues, less degradation of the tea catechins .md a high retention of the tea flavors[31 l . .2. 4. Microorganism and enzymatic hydrolysis Some microorganisms isolated from the environment can degrade the caffeine , for example Stemphyllium 32 33 34 p. [ l , Penicillium sp. and Aspergillus sp. [ ' ] . Aspergillus, Penicillium and Rhizopus can degrade the caffeine in offee pulp. It is assumed that the degradation of caffeine in fungi takes place via the demethylation route, with :heophylline as the first formed intermediate. In this respect, the caffeine degradation pathway in filamentous fungi losely resembles that of . Under aerobic conditions, bacteria and fungi decompose caffeine of nucleotide recursors: a. Fungi occurs metabolic pathways that caffeine degrades to . And these ·ecomposition pathways are alike in plants. b. Bacterial decomposes caffeine initially produced , .lltimately into xanthine. And the key to this catabolism is demethylase. c. Some bacteria oxidize caffeine to uric cid first like Rhodococcus and Klebsiella, then the uric acid continues the purine metabolic pathways. d. Caffeine an also indirectly be decomposed by xanthine oxidase. Compared with the aerobic degradation, the decomposition 'egree of caffeine is incomplete and the period is longer under anaerobic conditions. Application of microbial degradation of caffeine : Some bacteria use the coffee husk into solid state :'ermentation, so the nutritional value of cattle and other livestock 1 s feed can be improved. The research conducted y Gummadi et al. indicates that caffeine demethylase production kinetics was growth-related. Caffeine degradation by microbes or microbial enzymes is effective in producing useful by-products besides ffering advantages in decaffeination. Theophylline, a metabolite of caffeine degradation being an adenosine .mtagonist, has been shown to reduce the incidence of contrast-induced nephropathy, is considered as an alternative .n the treatment of chronic obtrusive pulmonary disease . .2. 5. Breeding new cultivars with low caffeine content Cultivating low caffeine tea cultivars is the most effective way to produce low caffeine tea, and excellent

~ermplasm resource is an important material basis for genetic technology research and cultivars . In nhina, low caffeine resources are very abundant, such as Daba tea (caffeine content about 0. 07% ) , }in chang plant tea tree (caffeine content about 0. 06%) , Yanjin niuzhai tea (caffeine content < 1. 0%) and Houzhou tea caffeine content < 1. 0% ) , etc. 2. 5. 1. Conventinal crossbreeding breeding low caffeine tea cultivars The most economical, safest and effective method to take off the caffeine is conventional crossbreeding. venerally, it is believed that tea caffeine content less than 2% can be considered low caffeine-specific resources[Js ] . In Japan, they got 8 plant offsprings whose caffeine content are less than 1. 7% by using a low caffeine content >ariety hybridizing with Y abukita since 1989 and the lowest is 1. 42% . Recently, Tea Research Institute of Guangdong Academy of Agricultural Sciences cooperated with Sun Yat-sen ..miversity and they used wild nankunshan primary tea as a parent to hybrid breeding a number of cocoa new lines, mcluding "the 1st cocoa" and "the 2nd cocoa". The suitable system results of Cocoa tea for black tea, green tea, ..-ellow tea and showed that its inclusions are richful and it has sucrose incense , aroma and strong ".lith slightly bitter. It may come to the conclusion that cocoa is capable of being domesticated as a home-grown tea and it can be developed into a natural non-caffeine beverage different from traditional tea. 424 "f" Journal of Tea 39 4-

2. 5. 2. Genetic engineering reduced tea caffeine Currently the pathway about the biosynthesis of caffeine m tea has been basically clear, and the full-length eDNA sequences of the three key genes ( SAM synthetase, inosine monophosphate dehydrogenase and gene) have also been cloned. Therefore, the most convenient way to reduce the caffeine in tea is that using antisense RNA technology or RNAi inhibits the key gene expression in caffeine biosynthesis. But the Camellia sinensis is less sensitive to Agrobacterium, so conversion rate is very low and genetic transformation techniques in tea have been a world problem stuck in vector construction and transformation of post-resistant callus 36 39 1 40 41 1 screening stage[ - until Indian scientists Mohanpuria et al. [ ' introduced the RNAi vectors containing caffeine synthase gene fragment into tea cotyledon callus and the root of tea tree seedings germinating a month through particle bombardment and Agrobacterium method, and obtained respectively 11 and 7 of transgenic plants contained the vected successfully. The caffeine and theobromine in these plants are respectively 44%-61% and 46% -67% lower than control group. And the result not only confirms the possibility of genetic engineering technology to reducing caffeine in tea, but also firstly confirms caffeine synthase synthesis of both caffeine and theobromine. However, tea is a natural cross-pollination of plants with self-incompatibility characteristics, which is not necessary to pass the parents' genetic traits, because the transgenic plants is heterozygous using its cotyledon induced callus to transform or tea tree seedling roots from seed germination by direct conversion. Therefore, it remains to be established an efficient genetic transformation system, using the tea tree stems, leaves and roots, and other stable genetic characteristics of the parent organization as explants. Through the system, a genetically modified tea reducing the caffeine content, and maintaining good genetic traits from parents can be found. 3. Conclusions The hot water treatment is a safe and inexpensive method for decaffeinating green tea, however, it seems only suitable for removal caffeine from fresh leaves. Some reports showed that DME have many advantages over other organic solvents. Having preliminary explored the laboratory and small-scale industrial production of the decaffeination using DME, the efficiency of the industrial mass decaffeinated using DME requires further study. The studies of SFE-C02 are more, and the technology is relatively mature, industrial production is feasible. Caffeine degradation by microbes or microbial enzymes is effective in producing useful by-products besides offering advantages in decaffeination. The demethylase and oxidases are involved in the microbial decaffeination process. Results showed purified demethylase is very unstable. Therefore future work pertaining to caffeine and its degradation can concentrate on the purification and characterisation of this enzyme. In the cultivation process, there are many issues that need further researches to clarify_ Such as , the caffeine in tea ( Camellia sinensis) is mainly synthesized in the chloroplast, and then transported to the vacuole, and combines with chlorogenic acid to form complexes , which gatheres in cell vacuole , but the transport mechanism is unclear; tea caffeine contents vary with cultivars, season, climate and cultivation measurements, and in different cells, tissues and organs, the factors which will affect the synthesis and decomposition of caffeine need to be further studied.

Acknowledgements This study was financed by the National Natural Science Foundation of China ( 31170644) and Tea industry & technology innovation strategic alliance foundation of Province (Project No. 7) .

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